Methyl 2-((allyloxy)methyl)acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation (in vitro) - Negative with and without metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Jul 2008 - 25 Aug 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Ministry of Labor Ordinance No. 76, 1988 and Ordinance No.13, 2000

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose-finding study:
Test substance groups: Selected in accordance with the “Guidelines”.
Positive control groups: Selected in accordance with the concentrations for positive control substances in “Guide to Mutagenicity Studies under the Industrial Safety and Health Act”
Concentrations: 0.00305, 0.0122, 0.00488, 0.195, 0781, 3.13, 12.5 and 50 mg/mL.

Main study:
In the dose-finding study, growth inhibitions were observed at 5000μg/plate for five tester strains in the absence and presence of the metabolic activity system. Therefore, the composition of groups in the main study included 6 dose levels by a common ratio of 2 both in the absence and presence of the metabolic activity system; from the highest dose of 5000 μg/plate to the lowest dose of 156 μg/plate.
Concentrations: 1.56, 3.13, 6.25, 12.5 and 50 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance is insoluble in water, but soluble at 50 mg/mL or more in DMSO. Therefore, DMSO was selected as the solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Additive volume of the prepared solution 100 μL per plate. Reason for the additive volume of the prepared solution 100 µL, which is the maximal possible volume without inhibition of bacterial growth by DMSO, was selected for positive control substances. Also, the same volume was selected for the test substance and negative control solutions.

45 μL of each tester strain were directly inoculated into 20 mL of 2.5% nutrient broth. The resulting mixture was cultured with shaking at 50 rpm for 9 hr at 37°C.
After incubation, the optical density of bacterial suspensions was measured to confirm that the viable bacterial count was not less than 1.0 × 10E9/mL.

In the absence of the metabolic activation system (without S9 mix):
500 μL of 100 mM Na-phosphate buffer (pH 7.4) and 100 μL of bacterial suspension were added to 100 μL of the test solution in each culture tube and the suspending solution was pre-incubated with shaking for 20 minutes at 37°C. Subsequently, 2 mL of a mixture of 0.5 mM biotin-0.5 mM histidine solution (in case of E. coli: 0.5 mM tryptophan solution) and 0.6% soft agar fluid (volume ratio 1:10) was added to each of the suspending solutions, and then poured onto the minimal glucose agar plate (Tesmedia AN Medium, Oriental Yeast Co., Ltd.,). All the plates were incubated for 48 hours at 37°C.
In the presence of metabolic activation system (With S9 mix):
500 µL of S9 mix and 100 µL of bacterial suspension were added to 100 µL of the test solution in each culture tube and the suspending solution was pre-incubated with shaking for 20 minutes at 37°C. Subsequently, 2 mL of a mixture of 0.5 mM biotin-0.5 mM histidine solution (in case of E. coli: 0.5 mM tryptophan solution) and 0.6% soft agar fluid (volume ratio 1:10) was added to each of the suspending solutions, and then poured onto the minimal glucose agar plate. All the plates were incubated for 48 hours at 37°C.

After incubation, the presence or absence of precipitation was checked visually for all dose levels. Moreover, the background lawn was observed using a microscope to confirm the presence or absence of bacterial growth inhibition. Thereafter, the numbers of revertant colonies were counted, twice for each plate, using a colony counter (PC-10NII, Toyo Sokki Co., Ltd.). The number of colonies was corrected and the mean of the corrected values was the number of revertant colonies for each plate.

Rationale for test conditions:

Guidelines

Evaluation criteria:

Acceptance criteria
From the number of revertant colonies on each plate, the mean values (rounded to integers) are obtained. If all of the following criteria are met, the results are considered positive.
1) The number of revertant colonies in the test substance group is equal to or greater than twice the mean negative control value.
2) Dose-dependency is observed.
3) Reproducibility is observed when comparing the dose-finding study and main study results.

Statistics:

not specified

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

For five tester strains, the number of revertant colonies in the test substance groups did not increase greater than twice that in the negative control group in the absence or presence of the metabolic activation system.
Bacterial growth inhibition was observed at 2500 µg/plate and more for five tester strains in the absence and presence of the metabolic activation system.
Precipitation of the test substance on the plates was not observed in five tester strains in the absence or presence of the metabolic activation system.

Conclusions:

In the results of the dose-finding study and main study, Methyl α-(Allyloxymethyl)acrylate did not induce increases in the number of revertant colonies for five tester strains in the absence or presence of the metabolic activation system, and the number did not reach more than twice that of the negative control group.
Therefore, Methyl α-(Allyloxymethyl)acrylate was judged not to be mutagenic agent, under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A Bacterial reverse mutation test was conducted (NISSEI BILIS Co. Ltd Shiga laboratory, 2008, Study 6297) to examine the potential for 2-(Allyloxymethyl) acrylic acid methyl ester to cause gene mutation.  The study was conducted according to Ministry of Labour Ordinance No. 76, 1988 and Ordinance No.13, 2000. The study was conducted in compliance with GLP.

The strains of Salmonella Typhimurium (TA1535, TA1537, TA98, and TA100) and Escherichia Coli (WP2 uvrA pKM101) were exposed to 2-(Allyloxymethyl) acrylic acid methyl ester as a solution in Dimethylsulphoxide (DMSO) in both the presence and absence of metabolic activation.

2-(Allyloxymethyl) acrylic acid methyl ester did not show significant signs of cytotoxicity at any of the levels tested and showed no signs of mutagenic activity in any of the tester strains used, either in the presence or the absence of metabolic activation.

 

Cytogenicity (mammalian cells) – not required at the registrant's tonnage band 

Gene mutation (mammalian cells) – not required at the registrant's tonnage band 

Short description of key information:

Bacterial reverse mutation (in vitro) - Negative with and without metabolic activation.

Chromosome aberration (in vitro) - no study available

Mouse micronucleus assay (in vivo) - no study available

 

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The study conducted did not indicate any evidence of mutagenic activity. On this basis it is concluded that there is no basis on which to classify 2-(Allyloxymethyl) acrylic acid methyl ester for mutagenic effects.